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FasL pathway
BBRC Biochemical and Biophysical Research Communications 310 (2003) 355–359 www.elsevier.com/locate/ybbrc
Shear stress induces apoptosis in vascular smooth muscle cells via an autocrine Fas/FasL pathway S. Apenberg, M.A. Freyberg, and P. Friedl* Technische Universit€ at Darmstadt, CSI f u€r. Org.Chemie and Biochemie, Petersenstr. 22, Darmstadt D-64287, Germany Received 3 September 2003
Abstract Endothelial lesions may lead to the exposure of vascular smooth muscle cells (VSMCs) to the blood flow. In such circumstances VSMCs are exposed to shear stress, an extraordinary mechanical stimulus for this type of cells. Rat VSMCs are cultivated in normal tissue culture plates (statically) or in a cone-plate viscometer (dynamically). Dynamic cultivation leads to a great increase of apoptosis. Immunofluorescence reveals the shear-stress-dependent expression of fas. Apoptosis can be induced by addition of fas ligand—a process which can be blocked by antibodies against either fas or fas ligand. Conditioned medium of dynamically cultivated VSMCs contains fas ligand as the only active apoptosis inducing activity. Apoptosis can be blocked by caspase inhibitors. So the exposure of VSMCs to shear stress leads to apoptosis by the establishment of an autocrine loop of fas and fas ligand—a potential mechanism for the prevention of narrowing of vessel diameter by VSMC proliferation. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Vascular smooth muscle cells; Shear stress; Fas; Mechanoregulation
It is well known that in response to a variety of different mechanical and chemical stimuli, cells can initiate highly conserved signalling events, which lead to either apoptosis or cell proliferation. For vascular smooth muscle cells (VSMCs) within atherosclerotic plaques and restenotic lesions these processes play a key role in vascular integrity and lesion formation. Apoptosis is a regulated “programmed” form of cell death and is characterized by a number of specific biochemical and morphological changes, including chromatin condensation, cytoplasmic condensation, membrane blebbing, and internucleosomal fragmentation of DNA [1,2]. Fas (also called APO-1 or CD95) is a type I glycosylated 45-kDa transmembrane protein that belongs to the tumor necrosis factor receptor famliy and transmits a suicide signal to the cell upon binding of its ligand (FasL), a highly conserved, 40-kDa glycoprotein [3]. In an initial step, binding of FasL leads to clustering of Fas followed by the activation of caspase-8/FLICE which in turn cleaves the inactive 32 kDa form of caspase-3/ * Corresponding author. Fax: +49-6151-164759. E-mail address: [email protected] (P. Friedl).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.025
CPP32 into two mature subunits of 17 and 12 kDa [4]. Active caspase-3/Cpp32 cleaves nuclear mitotic apparatus protein and mediates DNA fragmentation, chromatin condensation, and the formation of apoptotic bodies [5]. Many cell types express Fas whereas the expression of FasL is much more restricted. FasL is predominantly expressed in immune cells such as activated T cells, natural killer (NK) cells, and macrophages [6–9]. Both FasL and Fas are expressed in arterial tissue, including human atherosclerotic plaques. Immunohistochemical analyses revealed FasL expression in 34 of 34 carotid atherosclerotic plaques examined, with virtually all staining associated with intimal smooth muscle cells (SMCs) and only little staining in normal arterial tissue [10–12]. Fas is highly expressed in intimal SMCs of the plaque [10,12]. Fas/FasL expression and apoptosis in normal artery and plaque has prompted speculation on the role of these mediators in vascular cells [13,14]. In undiseased tissue apoptosis may inhibit arterial thickening by limiting cell proliferation and accumulation in the intima [10]. In atherosclerotic tissue, apoptosis of VSMCs may substantially weaken the plaque causing its
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S. Apenberg et al. / Biochemical and Biophysical Research Communications 310 (2003) 355–359
rupture, initiate thrombosis, and trigger acute coronary syndromes [14,15]. Despite clear evidence for FasL and Fas expression in VSMCs of the artery wall, the cellular mechanisms mediating FasL production in vascular cells are presently unknown. So, here, we investigate the role of mechanical stimuli in the fas system of VSMCs subjecting the cells to shear stress which is an unphysiological stimulus for VSMCs, as these cells are normally separated from blood flow by the endothelium. Apoptosis of VSMCs may prevent arterial thickening by limiting the number of VSMCs in injured vessels.
fine granules, brightly stained with DAPI throughout the entire nucleus and are counted as apoptotic. Several arbitrarily chosen visual fields are evaluated until at least 1000 nuclei have been counted for obtaining the percentage of apoptotic cells.
Materials and methods
Results
Materials
Apoptosis in VSMCs under dynamic cultivation
Dulbecco’s modified Eagle’s medium (DMEM), supplements, and trypsin/EDTA solution were purchased from Life Technologies (Eggenstein, Germany). Fetal bovine serum (FBS) was purchased from Seromed (Berlin, Germany). Rat VSMCs were a kind gift of Dr. D. Sedding (Giessen, Germany). DAPI (40 ,6-diamidino-2-phenylindole) was obtained from Sigma (Deisenhofen, Germany). Antibodies against Apo-1 SN were a kind gift of P.H. Krammer, German Cancer Research Center, Heidelberg. Anti-FasL antibody was obtained from BD Biosciences (Heidelberg/Germany), mouse gamma globulin, Cy3-conjugated streptavidin, and Cy3-conjugated F(ab)2 fragment (1.5 mg/ml) were from Dianova (Hamburg, Germany), FasL-peptide was from Biomol (Baesweiler/Germany), and caspase inhibitors were from Calbiochem (Bad Soden/Germany). Tissue culture plates, flasks, and ELISA-plates were purchased from Greiner (Frickenhausen, Germany). All chemicals used are of analytical grade. Methods Cell cultures The SMCs are cultivated in DMEM supplemented with 10% (v/v) FBS and 2 mM L -glutamine at 37 °C in a humidified incubator with 10% (v/v) CO2 . SMCs are detached from monolayer culture with trypsin/EDTA solution (0.5 g/L trypsin and 0.2 g/L EDTA in PBS) followed by DMEM containing 10% (v/v) FBS. The cells are reseeded at a ratio of 1:2 to 1:4. Dynamic cultivation and application of shear stress The SMCs are seeded in tissue culture dishes (94 16 or 35 10 mm) coated with 1% (w/v) gelatin. Two days after reaching confluence, the cells are starved in serumfree medium for 48 h. The cells are subjected to shear stress in a coneplate viscometer [16,17] for further 24–48 h. Application of stress is carried out at 37 °C in a humidified incubator with 10% (v/v) CO2 for the period indicated in the different tests. As static controls cultures of SMCs are seeded in culture dishes with the same diameter without application of shear stress. Apoptosis assay [18] The supernatant of the culture dish is carefully aspirated and the SMCs are immediately fixed with 0.1 ml 4% (w/v) formalin solution/ cm2 on ice for 20 min. The dish is washed once of with PBS, the DNA is stained for 15 min by addition of 2 lg/ml DAPI in methanol, and after a final wash with PBS the nuclear morphology is evaluated by fluorescence microscopy with a 20 objective. Fragmented nuclei show
Immunological staining for fas-receptor and CD 47 The fas-receptor and the CD 47 are visualized by immunostaining as described previously [19]. Preparation of conditioned medium from cultures under laminar flow The SMCs are seeded in tissue culture dishes (94 16) coated with 1% (w/v) gelatin. Two days after reaching confluence the cells are starved in serum-free medium for 48 h and then the cells are subjected to laminar shear stress in a cone-plate viscometer for further 24 h. The medium was collected and stored at )20 °C prior to use.
In a first set of experiments the influence of shear stress on VSMCs apoptosis shall be investigated. VSMCs are grown to confluence in 35-mm tissue culture dishes under static conditions. Two days after reaching confluence, the cells are starved in serum-free medium for another 2 days and then cultivated for 24 or 48 h under dynamic conditions. Table 1 shows that the amount of apoptotic cells in postconfluent monolayer cultures of rat VSMCs is affected directly by the influence of mechanical stress. Uniform laminar flow as well as turbulence lead to a significant increase of apoptosis. VSMCs express a fas-receptor under dynamic conditions In the next step we looked for transducing mediators linking mechanical stresses and apoptosis. So we compared the expression level of the fas-receptor in VSMCs cultured under different hemodynamic conditions as described above. The cells are stained for fas-receptor by indirect immunofluorescence. Fig. 1 shows that the fasreceptor is expressed under static and dynamic conditions at a comparable intensity.
Table 1 Effect of different mechanical stimuli on apoptosis of VSMCs Culture conditions
Time of incubation
Apoptotic cells SEM (%)
Static Laminar flow Turbulent flow
48 48 48
0.6 0.2 5.2 1.0 3.8 0.7
The cells are grown to confluence in 35-mm tissue culture dishes. Two days after reaching confluence, the cells are starved in serum-free medium for another 2 days and then cultivated for 48 h in a cone-plate apparatus under static conditions and laminar as well as turbulent flow at 1 dyn/cm2 . The means SD of percentages apoptotic cells are shown.
S. Apenberg et al. / Biochemical and Biophysical Research Communications 310 (2003) 355–359
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Fig. 1. fas-staining of VCMC (A) statically or (B) dynamically (steady laminar flow) cultivated cultures of VSMCs (5 days after reaching confluence) stained with anti-fas antibody and Cy3-conjugated F(ab)2 -fragment. The negative controls are samples with mouse gamma globulin substituting the first antibody: (C) of statically (D) of dynamically cultivated cells. Pictures are taken from a Zeiss fluorescence microscope (40).
Functionality of the fas-receptor The fas-receptor is expressed at low levels under static and dynamic conditions raising the question if the receptor is functional and able to induce apoptosis. This question was answered by a further cultivation with or without addition of 20 ng/ml recombinant fas-ligand. Whereas control cultures show a basal apoptotic rate of 2.0 0.3%, the addition of FasL increases the apoptotic rate up to 21.3 1.6%. So the fas-receptor is definitively functional and can be addressed to induce apoptosis in VSMCs. Involvement of FasL in apoptosis of VSMCs The above result raises the question if the fas-ligand is expressed under dynamic culture conditions and raises the level of apoptosis in VSMCs under these conditions. The cells are grown to confluence in 35-mm tissue culture dishes. Two days after reaching confluence, the cells are starved in serum-free medium for another 2 days and are further cultivated in dynamically or statically (control) conditioned culture medium with or without addition of 20 ng/ml fas-ligand antibody or with or without 10 lg/ml fas-ligand neutralizing antibody. Fig. 2 shows that dynamically conditioned culture medium increases the level of apoptosis between 10- and 11-fold compared to cells cultured in statically conditioned medium with 2.7 0.5% apoptosis (control). This effect can either be inhibited by addition of fas-ligand antibody or by addition of fas antibody.
Fig. 2. Effect of medium components on fas-dependent apoptosis of VSMCs. The cells are grown to confluence in 35-mm tissue culture dishes. Two days after reaching confluence, the cells are starved in serum-free medium for another 2 days and then cultivated in dynamically conditioned culture medium with or without addition of 20 ng/ml fas-ligand antibody or with or without 10 lg/ml fas-ligand neutralizing antibody. The amount of apoptotic cells was determined as described above.
Effect of caspase inhibitors on fas-ligand induced apoptosis of VSMCs We tested the effect of caspase inhibitors on fas-ligand induced apoptosis of VSMCs. The VSMCs are grown to confluence in 35-mm tissue culture dishes. Two days after reaching confluence the cells are starved in serum-free medium for another 2 days and then
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Table 2 Effect of caspase inhibitors on fas transduced apoptosis in VSMCs Inhibitor
Culture medium
Apoptotic cells SEM (%)
None None None Z-DEVD (caspase-3) Z-IETD (caspase-8) Z-LEHD (caspase-9)
Fresh Statically conditioned Dynamically conditioned Dynamically conditioned Dynamically conditioned Dynamically conditioned
1.6 0.2 2.7 0.5 14.7 1.2 1.8 0.4 1.6 0.3 6.7 0.8
The cells were incubated with or without dynamically conditioned medium for 48 h in the presence or absence of 100 lM of indicated inhibitors of caspases and subjected to apoptosis determination after DAPI staining. The means SD of percentages apoptotic cells are shown.
cultivated in statically or dynamically conditioned culture medium with different caspase inhibitors. Table 2 shows that dynamically conditioned culture medium leads to a 7-fold increase of apoptosis in VSMCs, which could be blocked when cultures were incubated with caspase inhibitors Z-DEVD and Z-IETD. Incubation with caspase inhibitor Z-LEHD reduced the level of apoptosis down to 50%.
Discussion Our results support the hypothesis that fas signalling and the regulation of fas-ligand expression play an important role for cellular survival of VSMCs. We have shown that enhanced levels of apoptosis occur in confluent VSMC cultures, cultivated under dynamic conditions where an unphysiological mechanical stress such as laminar or turbulent flow is exerted to VSMCs. Furthermore, we found weak expression of fas-receptor, however, also to a comparable amount under static conditions, where VSMCs do not undergo apoptosis even in the absence of serum-growth factors. The receptor was unequivocally proven to be functional by induction of massive apoptosis upon addition of recombinant fas-ligand. So the induction of apoptosis under dynamic culture conditions might involve the autocrine secretion of fas-ligand with successful binding to fas on the cell surface. Consequently dynamically conditioned culture medium induced massive apoptosis which could be blocked by adding neutralizing monoclonal antibodies against either fas or fasligand. These findings demonstrate for the first time that an autocrine loop of fas and fas-ligand is involved in apoptosis of vascular smooth muscle cells. This loop is physiologically senseful since it counteracts the proliferation of VSMCs exposed to blood flow due to lesions of the vascular endothelium. The lack of an endothelial barrier opens the VSMCs to the action of mitogens originating from blood or blood components as, for example, PDGF. An uncontrolled or insufficiently controlled proliferation will directly lead to a narrowing of the vessel diameter with consequent pathophysiological situations. The shear stress sensitive
expression of fas/fasL described here is another example for the mechanoregulation of an autocrine loop equivalent to the previously described thrombospondin/CD47 loop of endothelial cells [20,21]. We assume that this is a general mechanism and expect that in future more examples of this kind will be described. To further investigate the role of caspases in the fas/ fas-ligand apoptosis pathway, we tested whether inhibition of caspase 3, 8 or 9 by caspase inhibitors reduces the levels of apoptosis. The inhibitors of caspase 3 and 8 prevent the increase of apoptosis and keep the level of apoposis even below the basic level of static cultivation. This is in accordance with the idea of caspase 8 being the unique Fas-receptor caspase necessary for initiation of intracellular signalling and caspase 3 being an essential downstream caspase at the end of caspase cascades [22]. Caspase 9 inhibitor reduces the level of apoptosis by 45–55%, indicating that about half of the caspase 8 induced signalling involves the mitochondrion-associated pathway [23]. In addition to the here-shown fas pathway of VSMCs, other death pathways may operate in atherosclerotic lesions. For example, activated macrophages may exert cytotoxic stress leading to induction of cell death [10]. It has also been shown that vascular endothelial cells express functional fas-receptors due to lack of a steady laminar flow [19]. Both the presence of macrophages and the lack of an uniform laminar flow are characteristics of atherosclerotic plaques. In conclusion, this paper elucidates for the first time VSMC apoptosis via the fas/fasL pathway as the result of a shear stress acting on VSMCs hinting at a regulatory role of this process for the maintenance of a functional vessel topology.
References [1] A. Ashkenazi, V.M. Dixit, Science 281 (1998) 1305–1308. [2] A. Ashkenazi, V.M. Dixit, Curr. Opin. Cell Biol. 11 (1999) 255– 260. [3] S. Nagata, Cell 88 (1997) 355–365. [4] P. Juo, C.J. Kuo, J. Yuan, J. Blenis, Curr. Biol. 8 (1998) 1001–1008.
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[14] P.D. Flynn, C.D. Byrne, T.P. Baglin, P.L. Weissberg, M.R. Bennett, Blood 89 (1997) 4378–4384. [15] M.M. Kockx, A.G. Herman, Cardiovasc. Res. 45 (2000) 736–746. [16] H.P. Sdougos, S.R. Bussolari, C.F. Dewey Jr., J. Fluid. Mech. 138 (1984) 379–404. [17] R. Graf, S. Apenberg, M. Freyberg, P. Friedl, Apoptosis (2003), in press. [18] D. Kaiser, M.A. Freyberg, G. vonWichert, P. Marenbach, H. Tolle, P. Friedl, in: O.W. Merten (Ed.), Animal Cell Technology: New Developments—New Applications, Kluwer Academic Publishers, Dordrecht, 1998, pp. 729–731. [19] M.A. Freyberg, D. Kaiser, R. Graf, P. Friedl, Apoptosis 6 (2001) 339–343. [20] M.A. Freyberg, D. Kaiser, R. Graf, P. Vischer, P. Friedl, Biochem. Biophys. Res. Commun. 271 (2000) 584–588. [21] M.A. Freyberg, D. Kaiser, R. Graf, J. Buttenbender, P. Friedl, Biochem. Biophys. Res. Commun. 286 (2001) 141–149. [22] T. Suhara, H.S. Kim, L.A. Kirshenbaum, K. Walsh, Mol. Cell. Biol. 22 (2002) 680–691. [23] M.O. Hengartner, Nature 407 (2000) 770–776.
Shear stress induces apoptosis in vascular smooth muscle cells via an autocrine Fas/FasL pathway S. Apenberg, M.A. Freyberg, and P. Friedl* Technische Universit€ at Darmstadt, CSI f u€r. Org.Chemie and Biochemie, Petersenstr. 22, Darmstadt D-64287, Germany Received 3 September 2003
Abstract Endothelial lesions may lead to the exposure of vascular smooth muscle cells (VSMCs) to the blood flow. In such circumstances VSMCs are exposed to shear stress, an extraordinary mechanical stimulus for this type of cells. Rat VSMCs are cultivated in normal tissue culture plates (statically) or in a cone-plate viscometer (dynamically). Dynamic cultivation leads to a great increase of apoptosis. Immunofluorescence reveals the shear-stress-dependent expression of fas. Apoptosis can be induced by addition of fas ligand—a process which can be blocked by antibodies against either fas or fas ligand. Conditioned medium of dynamically cultivated VSMCs contains fas ligand as the only active apoptosis inducing activity. Apoptosis can be blocked by caspase inhibitors. So the exposure of VSMCs to shear stress leads to apoptosis by the establishment of an autocrine loop of fas and fas ligand—a potential mechanism for the prevention of narrowing of vessel diameter by VSMC proliferation. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Vascular smooth muscle cells; Shear stress; Fas; Mechanoregulation
It is well known that in response to a variety of different mechanical and chemical stimuli, cells can initiate highly conserved signalling events, which lead to either apoptosis or cell proliferation. For vascular smooth muscle cells (VSMCs) within atherosclerotic plaques and restenotic lesions these processes play a key role in vascular integrity and lesion formation. Apoptosis is a regulated “programmed” form of cell death and is characterized by a number of specific biochemical and morphological changes, including chromatin condensation, cytoplasmic condensation, membrane blebbing, and internucleosomal fragmentation of DNA [1,2]. Fas (also called APO-1 or CD95) is a type I glycosylated 45-kDa transmembrane protein that belongs to the tumor necrosis factor receptor famliy and transmits a suicide signal to the cell upon binding of its ligand (FasL), a highly conserved, 40-kDa glycoprotein [3]. In an initial step, binding of FasL leads to clustering of Fas followed by the activation of caspase-8/FLICE which in turn cleaves the inactive 32 kDa form of caspase-3/ * Corresponding author. Fax: +49-6151-164759. E-mail address: [email protected] (P. Friedl).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.09.025
CPP32 into two mature subunits of 17 and 12 kDa [4]. Active caspase-3/Cpp32 cleaves nuclear mitotic apparatus protein and mediates DNA fragmentation, chromatin condensation, and the formation of apoptotic bodies [5]. Many cell types express Fas whereas the expression of FasL is much more restricted. FasL is predominantly expressed in immune cells such as activated T cells, natural killer (NK) cells, and macrophages [6–9]. Both FasL and Fas are expressed in arterial tissue, including human atherosclerotic plaques. Immunohistochemical analyses revealed FasL expression in 34 of 34 carotid atherosclerotic plaques examined, with virtually all staining associated with intimal smooth muscle cells (SMCs) and only little staining in normal arterial tissue [10–12]. Fas is highly expressed in intimal SMCs of the plaque [10,12]. Fas/FasL expression and apoptosis in normal artery and plaque has prompted speculation on the role of these mediators in vascular cells [13,14]. In undiseased tissue apoptosis may inhibit arterial thickening by limiting cell proliferation and accumulation in the intima [10]. In atherosclerotic tissue, apoptosis of VSMCs may substantially weaken the plaque causing its
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rupture, initiate thrombosis, and trigger acute coronary syndromes [14,15]. Despite clear evidence for FasL and Fas expression in VSMCs of the artery wall, the cellular mechanisms mediating FasL production in vascular cells are presently unknown. So, here, we investigate the role of mechanical stimuli in the fas system of VSMCs subjecting the cells to shear stress which is an unphysiological stimulus for VSMCs, as these cells are normally separated from blood flow by the endothelium. Apoptosis of VSMCs may prevent arterial thickening by limiting the number of VSMCs in injured vessels.
fine granules, brightly stained with DAPI throughout the entire nucleus and are counted as apoptotic. Several arbitrarily chosen visual fields are evaluated until at least 1000 nuclei have been counted for obtaining the percentage of apoptotic cells.
Materials and methods
Results
Materials
Apoptosis in VSMCs under dynamic cultivation
Dulbecco’s modified Eagle’s medium (DMEM), supplements, and trypsin/EDTA solution were purchased from Life Technologies (Eggenstein, Germany). Fetal bovine serum (FBS) was purchased from Seromed (Berlin, Germany). Rat VSMCs were a kind gift of Dr. D. Sedding (Giessen, Germany). DAPI (40 ,6-diamidino-2-phenylindole) was obtained from Sigma (Deisenhofen, Germany). Antibodies against Apo-1 SN were a kind gift of P.H. Krammer, German Cancer Research Center, Heidelberg. Anti-FasL antibody was obtained from BD Biosciences (Heidelberg/Germany), mouse gamma globulin, Cy3-conjugated streptavidin, and Cy3-conjugated F(ab)2 fragment (1.5 mg/ml) were from Dianova (Hamburg, Germany), FasL-peptide was from Biomol (Baesweiler/Germany), and caspase inhibitors were from Calbiochem (Bad Soden/Germany). Tissue culture plates, flasks, and ELISA-plates were purchased from Greiner (Frickenhausen, Germany). All chemicals used are of analytical grade. Methods Cell cultures The SMCs are cultivated in DMEM supplemented with 10% (v/v) FBS and 2 mM L -glutamine at 37 °C in a humidified incubator with 10% (v/v) CO2 . SMCs are detached from monolayer culture with trypsin/EDTA solution (0.5 g/L trypsin and 0.2 g/L EDTA in PBS) followed by DMEM containing 10% (v/v) FBS. The cells are reseeded at a ratio of 1:2 to 1:4. Dynamic cultivation and application of shear stress The SMCs are seeded in tissue culture dishes (94 16 or 35 10 mm) coated with 1% (w/v) gelatin. Two days after reaching confluence, the cells are starved in serumfree medium for 48 h. The cells are subjected to shear stress in a coneplate viscometer [16,17] for further 24–48 h. Application of stress is carried out at 37 °C in a humidified incubator with 10% (v/v) CO2 for the period indicated in the different tests. As static controls cultures of SMCs are seeded in culture dishes with the same diameter without application of shear stress. Apoptosis assay [18] The supernatant of the culture dish is carefully aspirated and the SMCs are immediately fixed with 0.1 ml 4% (w/v) formalin solution/ cm2 on ice for 20 min. The dish is washed once of with PBS, the DNA is stained for 15 min by addition of 2 lg/ml DAPI in methanol, and after a final wash with PBS the nuclear morphology is evaluated by fluorescence microscopy with a 20 objective. Fragmented nuclei show
Immunological staining for fas-receptor and CD 47 The fas-receptor and the CD 47 are visualized by immunostaining as described previously [19]. Preparation of conditioned medium from cultures under laminar flow The SMCs are seeded in tissue culture dishes (94 16) coated with 1% (w/v) gelatin. Two days after reaching confluence the cells are starved in serum-free medium for 48 h and then the cells are subjected to laminar shear stress in a cone-plate viscometer for further 24 h. The medium was collected and stored at )20 °C prior to use.
In a first set of experiments the influence of shear stress on VSMCs apoptosis shall be investigated. VSMCs are grown to confluence in 35-mm tissue culture dishes under static conditions. Two days after reaching confluence, the cells are starved in serum-free medium for another 2 days and then cultivated for 24 or 48 h under dynamic conditions. Table 1 shows that the amount of apoptotic cells in postconfluent monolayer cultures of rat VSMCs is affected directly by the influence of mechanical stress. Uniform laminar flow as well as turbulence lead to a significant increase of apoptosis. VSMCs express a fas-receptor under dynamic conditions In the next step we looked for transducing mediators linking mechanical stresses and apoptosis. So we compared the expression level of the fas-receptor in VSMCs cultured under different hemodynamic conditions as described above. The cells are stained for fas-receptor by indirect immunofluorescence. Fig. 1 shows that the fasreceptor is expressed under static and dynamic conditions at a comparable intensity.
Table 1 Effect of different mechanical stimuli on apoptosis of VSMCs Culture conditions
Time of incubation
Apoptotic cells SEM (%)
Static Laminar flow Turbulent flow
48 48 48
0.6 0.2 5.2 1.0 3.8 0.7
The cells are grown to confluence in 35-mm tissue culture dishes. Two days after reaching confluence, the cells are starved in serum-free medium for another 2 days and then cultivated for 48 h in a cone-plate apparatus under static conditions and laminar as well as turbulent flow at 1 dyn/cm2 . The means SD of percentages apoptotic cells are shown.
S. Apenberg et al. / Biochemical and Biophysical Research Communications 310 (2003) 355–359
357
Fig. 1. fas-staining of VCMC (A) statically or (B) dynamically (steady laminar flow) cultivated cultures of VSMCs (5 days after reaching confluence) stained with anti-fas antibody and Cy3-conjugated F(ab)2 -fragment. The negative controls are samples with mouse gamma globulin substituting the first antibody: (C) of statically (D) of dynamically cultivated cells. Pictures are taken from a Zeiss fluorescence microscope (40).
Functionality of the fas-receptor The fas-receptor is expressed at low levels under static and dynamic conditions raising the question if the receptor is functional and able to induce apoptosis. This question was answered by a further cultivation with or without addition of 20 ng/ml recombinant fas-ligand. Whereas control cultures show a basal apoptotic rate of 2.0 0.3%, the addition of FasL increases the apoptotic rate up to 21.3 1.6%. So the fas-receptor is definitively functional and can be addressed to induce apoptosis in VSMCs. Involvement of FasL in apoptosis of VSMCs The above result raises the question if the fas-ligand is expressed under dynamic culture conditions and raises the level of apoptosis in VSMCs under these conditions. The cells are grown to confluence in 35-mm tissue culture dishes. Two days after reaching confluence, the cells are starved in serum-free medium for another 2 days and are further cultivated in dynamically or statically (control) conditioned culture medium with or without addition of 20 ng/ml fas-ligand antibody or with or without 10 lg/ml fas-ligand neutralizing antibody. Fig. 2 shows that dynamically conditioned culture medium increases the level of apoptosis between 10- and 11-fold compared to cells cultured in statically conditioned medium with 2.7 0.5% apoptosis (control). This effect can either be inhibited by addition of fas-ligand antibody or by addition of fas antibody.
Fig. 2. Effect of medium components on fas-dependent apoptosis of VSMCs. The cells are grown to confluence in 35-mm tissue culture dishes. Two days after reaching confluence, the cells are starved in serum-free medium for another 2 days and then cultivated in dynamically conditioned culture medium with or without addition of 20 ng/ml fas-ligand antibody or with or without 10 lg/ml fas-ligand neutralizing antibody. The amount of apoptotic cells was determined as described above.
Effect of caspase inhibitors on fas-ligand induced apoptosis of VSMCs We tested the effect of caspase inhibitors on fas-ligand induced apoptosis of VSMCs. The VSMCs are grown to confluence in 35-mm tissue culture dishes. Two days after reaching confluence the cells are starved in serum-free medium for another 2 days and then
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S. Apenberg et al. / Biochemical and Biophysical Research Communications 310 (2003) 355–359
Table 2 Effect of caspase inhibitors on fas transduced apoptosis in VSMCs Inhibitor
Culture medium
Apoptotic cells SEM (%)
None None None Z-DEVD (caspase-3) Z-IETD (caspase-8) Z-LEHD (caspase-9)
Fresh Statically conditioned Dynamically conditioned Dynamically conditioned Dynamically conditioned Dynamically conditioned
1.6 0.2 2.7 0.5 14.7 1.2 1.8 0.4 1.6 0.3 6.7 0.8
The cells were incubated with or without dynamically conditioned medium for 48 h in the presence or absence of 100 lM of indicated inhibitors of caspases and subjected to apoptosis determination after DAPI staining. The means SD of percentages apoptotic cells are shown.
cultivated in statically or dynamically conditioned culture medium with different caspase inhibitors. Table 2 shows that dynamically conditioned culture medium leads to a 7-fold increase of apoptosis in VSMCs, which could be blocked when cultures were incubated with caspase inhibitors Z-DEVD and Z-IETD. Incubation with caspase inhibitor Z-LEHD reduced the level of apoptosis down to 50%.
Discussion Our results support the hypothesis that fas signalling and the regulation of fas-ligand expression play an important role for cellular survival of VSMCs. We have shown that enhanced levels of apoptosis occur in confluent VSMC cultures, cultivated under dynamic conditions where an unphysiological mechanical stress such as laminar or turbulent flow is exerted to VSMCs. Furthermore, we found weak expression of fas-receptor, however, also to a comparable amount under static conditions, where VSMCs do not undergo apoptosis even in the absence of serum-growth factors. The receptor was unequivocally proven to be functional by induction of massive apoptosis upon addition of recombinant fas-ligand. So the induction of apoptosis under dynamic culture conditions might involve the autocrine secretion of fas-ligand with successful binding to fas on the cell surface. Consequently dynamically conditioned culture medium induced massive apoptosis which could be blocked by adding neutralizing monoclonal antibodies against either fas or fasligand. These findings demonstrate for the first time that an autocrine loop of fas and fas-ligand is involved in apoptosis of vascular smooth muscle cells. This loop is physiologically senseful since it counteracts the proliferation of VSMCs exposed to blood flow due to lesions of the vascular endothelium. The lack of an endothelial barrier opens the VSMCs to the action of mitogens originating from blood or blood components as, for example, PDGF. An uncontrolled or insufficiently controlled proliferation will directly lead to a narrowing of the vessel diameter with consequent pathophysiological situations. The shear stress sensitive
expression of fas/fasL described here is another example for the mechanoregulation of an autocrine loop equivalent to the previously described thrombospondin/CD47 loop of endothelial cells [20,21]. We assume that this is a general mechanism and expect that in future more examples of this kind will be described. To further investigate the role of caspases in the fas/ fas-ligand apoptosis pathway, we tested whether inhibition of caspase 3, 8 or 9 by caspase inhibitors reduces the levels of apoptosis. The inhibitors of caspase 3 and 8 prevent the increase of apoptosis and keep the level of apoposis even below the basic level of static cultivation. This is in accordance with the idea of caspase 8 being the unique Fas-receptor caspase necessary for initiation of intracellular signalling and caspase 3 being an essential downstream caspase at the end of caspase cascades [22]. Caspase 9 inhibitor reduces the level of apoptosis by 45–55%, indicating that about half of the caspase 8 induced signalling involves the mitochondrion-associated pathway [23]. In addition to the here-shown fas pathway of VSMCs, other death pathways may operate in atherosclerotic lesions. For example, activated macrophages may exert cytotoxic stress leading to induction of cell death [10]. It has also been shown that vascular endothelial cells express functional fas-receptors due to lack of a steady laminar flow [19]. Both the presence of macrophages and the lack of an uniform laminar flow are characteristics of atherosclerotic plaques. In conclusion, this paper elucidates for the first time VSMC apoptosis via the fas/fasL pathway as the result of a shear stress acting on VSMCs hinting at a regulatory role of this process for the maintenance of a functional vessel topology.
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